More than two dozen cytokines have been identified that regulate blood composition by controlling the growth and differentiation of hematopoietic stem cells. Interferons, tumor necrosis factor, stem cell factor, the numbered interleukins, ligands of oncogene receptors, and the various colony stimulating factors are exemplary of these proteins and glycoproteins. One of these factors, interleukin-6 (IL-6) was originally identified as a B-cell differentiation factor, but has subsequently been shown to induce acute phase protein synthesis in liver cells, to inhibit growth of certain myeloid leukemia cell lines and induce their differentiation into macrophage cells, to promote IL-3 dependent colony formation of primitive blast colony forming cells, to cause differentiation of neuronal cells, to enhance keratinocyte and mesangial cell growth, to promote the maturation of megakaryocytes, and to induce the proliferation and differentiation of T cells. In vivo, IL-6 increases the hematopoietic cell count of the erythroid, myeloid, and thrombocytic lineages. Other former names for IL-6 are .beta.2-interferon, B-cell stimulatory factor-2, hybridoma/plasmacytoma growth factor, and monocyte granulocyte inducer type 2. The spectrum of activities attributable to IL-6 indicates that it is useful in tumor inhibition, bone remodeling, kidney development, and T- and B-cell proliferation and stimulation.
Interleukin-11 has been shown to augment hematopoietic proliferation and differentiation of cells from normal mice, increase B-cell maturation, augment macrphage proliferation and megakaryocyte maturation, proliferate multipotent hematopoietic progenitors, stimulate early murine progenitors, and inhibit adipogenesis. Burstein et al., Journal of Cellular Physiology (1992) 153:312; Bazan, Neuron (1991) 7:197; and Yang et al., BioFactors (1992) 4:15-21.
Ciliary neurotrophic factor (CNTF) receptor is most homologous to the IL-6 receptor and lacks a cytoplasmic domain. Except for skeletal muscle, peripheral (sciatic) nerve and adrenal gland, CNTF receptor expression appears confined to the central nervous system. CNTF has been shown to promote neuronal differentiation and neuron augmentation. Research results implicate CNTF in the trophic support of a broad range of peripheral and central neurons, broader in fact, than that of the neurotrophins. Lo, Proc. Natl. Acad. Sci. USA (1993) 90; 2557-2558. Specifically, CNTF has shown "rescue" effects on embryonic spinal cord motor neurons, axotomized facial motor neurons in young rats and degenerative motor neurons in mouse mutant progressive motor neuropathy. In vivo, CNTF infused into the lateral ventrical of fimbria-fornix-lesioned adult rats prevents degeneration of almost all septal neurons including many non-cholinergic neurons that are not maintained by nerve growth factor. Unsicker et al., Neurobiology (1992) 2:671-678.
Leukemia inhibitory factor (LIF) has been demonstrated to inhibit the growth of certain myeloid leukemia cells and to induce their differentiation into macrophage cells; to enhance interleukin-3 dependent colony formation of primitive blast cells; to promote megakaryocyte growth and differentiation; to induce neuronal differentiation; to stimulate the production of acute phase proteins and hepatocytes (all properties it shares with IL-6) and to inhibit the differentiation of embryonic stem cells and kidney cells and to induce bone resorption.
Oncostatin-M (OSM) is known to be a tumor inhibitor for melanoma and certain carcinoma cells and inhibits the growth of human A375 melanoma cells but not normal human fibroblasts. It is also an inhibitor of the growth of M1 myeloid leukemic cells and induces their differentiation into macrophage-like cells as well as stimulating megakaryocyte production in the spleen. OSM is also known to inhibit embryonic stem cell differentiation, induce hepatic cell acute-phase protein sythesis, induce mitosis of AIDS-related Kaposi's sarcoma cell and vascular smooth muscle cell and induce neuronal differentiation.
Granulocyte colony stimulating factor (G-CSF) stimulates neutrophil proliferation and differentiation and induces the differentiation of M1 murine myeloid leukemic cells into macrophage-like cells as well as enhancing interleukin-3 dependent colony formation of primitive blast cells. It appears to have little effect on the hematopoietic cell lineages of megakaryocytes or platelets but enhances cytosine arabinoside-mediated cytoxicity in human myeloid leukemia cells.
Reported biological activities of the foregoing cytokine family members are summarized in the following table:
TABLE I Reported Biological Activities of Cytokine Family Members LIF OSM G-CSF IL-6 L-11 CNTF Endothelial Cell Proliferation NR + NR NR NR NR Tumor Inhibition + + NR + NR NR Embryonic Stem Cell Maintenance + + NR NR NR NR Hematopoietic Leukemic Cell Differentiation + + + + + NR Melanoma Cell Inhibition - + - + NR NR Neutrophil Proliferation/Stimulation NR NR + + + NR Myoblast Proliferation + NR NR NR NR NR Bone Remodeling + NR NR + NR NR Kidney Development + NR NR NR NR NR Neuronal Differentiation + + NR + NR + Hepatocyte Stimulation + + NR + NR NR Megakaryocyte Augmentation + + - + + NR T-Cell Proliferation NR NR NR + NR NR Keratinocyte Proliferation NR NR NR + NR NR B-Cell Proliferation/Stimulation NR NR NR + + NR Binding to Human Placental Cell Receptor + + - - NR NR Hemopoietic Proliferation (Normal) NR NR NR + + NR Neuron Augmentation NR NR NR NR NR + "+" = activity; "-" = no activity; and "NR" = not reported.
As shown in the foregoing table, the six cytokines exhibit different activities. For example, OSM and IL-6 inhibit the growth of melanoma cells; LIF and G-CSF do not. However, LIF, IL-6 and IL-11 and G-CSF differ in that LIF, IL-6 and IL-11 are capable of stimulating proliferation and differentiation of megakaryocytes. OSM binds to human placental cell receptor; IL-6 does not; CNTF, IL-6, OSM and LIF stimulate neuronal differentiation, G-CSF may not.
Although some cytokines may enhance immune system health and white blood cell replacement in patients with depleted lymphocyte populations (e.g., patients undergoing radiation or chemotherapy) or exhibit other desireable biological activity, each cytokine discussed above has a structure unique for intended function(s). Biological mechanisms are known to be highly selective and responsive only when intricate signaling systems invoke particular biochemical responses at a cellular (biochemical) level. Thus, each cytokine targets a specific cell receptor, inducing a particular biological reaction, producing a specific biological result.
These cytokines, CNTF, G-CSF, IL-6, IL-11, LIF and OSM, utilize specific mechanisms for effecting identified cellular responses. Their structural and biochemical specificity do not suggest that alternative cytokines, radically different from these cytokines, would have unexpected advantages in selective biological mechanisms over a much broader range of biological activities. One attempt to provide a therapeutic compound with multiple cytokine function resulted in a fusion protein, PIXY321, having an entire granulocyte macrophage colony stimulating factor (GM-CSF) cytokine, a linking sequence and an entire interleukin-3 (IL-3) cytokine. The extremely large polypeptide (spanning more than two cytokines) has both GM-CSF and IL-3 activity yet is difficult to produce and administer.